Little consideration has been given to the possibility that certain phases of viral infectious cycles are regulated by the rapid and selective turnover of viral proteins. Such selective degradation may be a significant means of controlling viral protein levels in infected cells, and it may represent a previously unrecognized site for the development of antiviral therapies. The encephalomyocarditis (EMC) virus 3C protease is rapidly and selectively degraded, both in vivo in mouse cells and in vitro in reticulocyte lysate. This study will explore the proteolytic system responsible for the rapid degradation of the EMC 3C virus protease in reticulocyte lysate, with the idea that this may serve as a general model for the turnover of viral proteins. This project has three aims: 1) to determine if poliovirus 3C protease is also rapidly degraded in reticulocyte lysate, thus providing some indication of the uniqueness of the behavior of the EMC virus 3C protease; 2) to characterize the nature of the proteolytic system and to determine if the ubiquitin-mediated process is involved; and 3) to identify and isolate the components of the proteolytic system, with special emphases given to identifying the RNA species which is suspected of being required. Poliovirus 3C and EMC virus 3C proteases will be prepared from in vitro transcription/translation, or from E. coli cells expressing the cloned EMC virus 3C gene. The ability of whole and fractionated reticulocyte lysate to rapidly degrade these substrates in the presence of specific protease inhibitors and inhibitors of ubiquitination will be evaluated. The ability of specific species or types of RNA to stimulate degradation in lysate fractions will be tested, and it will be determined if specific tRNA-mediated modifications of the EMC virus 3C protease occurs. Classical protein and nucleic acid purification procedures will be used to isolate any previously unrecognized protein and RNA factors which are involved in the turnover of the EMC virus 3C protease.